Homeotic genes in Drosophila such as Ultrabithorax (Ubx) and Antennapedia (Antp) select and maintain the identity of specific body segments during development, and must therefore be deployed at specific body positions at particular times in development. A regulatory network of about 30 genes, which play early roles on defining the major body axis and partitioning the animal into segments, also govern the initial patterns of expression of the homeotics, and these patterns are subsequently refined by cross- regulatory interactions between the homeotics. Maintenance of the patterns requires the action of another set of genes, the Polycomb group, and perhaps autoregulatory activities of the homeotics. The long term goal of this research is to reconstruct homeotic gene regulation in vitro from purified components, in order to determine the biochemical mechanisms of the processes by which gene expression is initiated, refined, and maintained during development. Several regulatory interactions have been recapitulated in a Drosophila cell culture system. Using a cotransfection assay in which a plasmid which express one gene product is introduced into cultured cells along with a candidate target gene, cross-regulatory and autoregulatory activities of Antp and Ubx proteins have been demonstrated. Also, the regulatory activity of fushi tarazu protein, the product of a gene involved in segmentation, has been demonstrated. Purified Ubx protein binds with high affinity to sequences in and around Antp and Ubx promoters, suggesting that at least some of these regulatory effects may be direct and mediated by protein binding to transcriptional control regions. The goals of this project are to determine the mechanism of regulation of Antp and Ubx expression by an Ubx protein, using a combined in vivo and in vitro approach and to begin to explore other aspects of homeotic gene regulation in the cell culture system. Specifically, we plan to: 1. Identify and characterize in detail the cis-acting sites of regulation of Ubx protein in the Antp and Ubx promoter regions using the cotransfection assay, and determine the relationship of these sites to the in vitro binding sites of the protein and to the regulatory sites of Antp and fushi tarazu proteins. 2. Evaluate the function of the identified regulatory sites in the developing animal. 3. Establish an in vitro system to study regulation of Antp and Ubx expression by Ubx protein, and begin biochemical fractionation of the system. 4. Attempt to reconstruct the combinatorial control of homeotic gene expression by fushi tarazu and other segmentation genes in the cell culture system.